Grüneberg1 has directed attention to two properties
of inbred strains which should counsel caution in their use for some experimental
purposes

(1) They cannot be relied upon to remain constant in their
heritable properties with the passage of time, and may diverge quite rapidly
when split up into separate sub-strains and colonies. (2) They do not constitute
phenotypically uniform material, but may, on the contrary, be strikingly more
variable than the F1 hybrids between strains.

This second proposition, for which Livesay2 obtained
experimental evidence in 1930, calls into question the established practice
of using inbred material for the bio-assay of food factors, hormones, drugs,
poisons, pathogens, etc. In such experiments precision in the estimates, and
hence uniformity in the test organisms, is of the highest importance.

The characters measured in bio-assay are of the type usually
termed 'physiological', whereas the examples cited by Grüneberg all relate
to morphological variation. None the less the interpretation of the phenomenon
advanced by Robertson and Reeves3 and by Rasmuson4 would
imply that we are dealing with a general biological law which must be expected
to apply no less to 'physiological' than to 'morphological' characters. "The
more heterozygous individuals," write Robertson and Reeve, "will
carry a greater diversity of alleles, and these are likely to endow them with
a greater biochemical versatility in development. This will lead to ... a
reduced sensitivity to environmental variations, since there will be more
ways of overcoming the obstacles which such variations put in the way of normal
development."

Exp.

Stock

Gm. 'Nembutal'
per cent

Mean log
response time

Antilog
(min.)

Sums of
squares

Degrees of
freedom

Estimate of
variance

I

Inbred F1 hybrids "Mousery"

0.65
0.65
0.65

1.412
1.125
1.662

26
13
46

0.4896
0.1827
0.4853

13
8
13

0.0377
0.0228
0.0373

II

Inbred F1 hybrids "Mousery"

0.65
0.85
0.65

1.593
2.110
2.185

39
129
153

0.8888
0.2394
0.5891

9
16
13

0.0988
0.0150
0.0453

Combined

Inbred F1 hybrids "Mousery"

1.3784
0.4221
1.0744

22
24
26

0.0627
0.0176
0.0413

Two experiments on variation in a 'physiological' character
have, in fact, just been published. Yoon5 has found in mice that
F1 hybrids are less variable in time of vaginal opening than the parental
strains. Maynard Smith and Maynard Smith6 working with Drosophila
subobscura have found a decreased variability in rate of development
associated with heterozygosity for a marked chromosome.

We have recently conducted experiments with mice on the variation
of a character chosen to be of the type measured in bio-assay, namely, the
duration of narcosis following the intraperitoneal injection of 0.10 ml. per
gm, body-weight of a dilution of a proprietary preparation ('Nembutal') of
pentobarbital sodium.

We compared the variability in response of samples of three
stocks of mice: inbreds from the C57BL strain, F1 hybrids from C57BL
female x C3H crosses, and random-bred albinos supplied by "The
Mousery", Rayleigh, Essex. Unfortunately, it was not possible to include
a sample of the other parental strain (C3H). The duration of narcosis
was expressed as the logarithm of the number of minutes elapsing from the
abolition to the reappearance of the righting reflex. The logarithmic transformation
was adopted in order to eliminate dependence of the variance upon the mean,
and was found to be satisfactory for this purpose.

There were indications of differences in response both between
the sexes and among different litters. In order to eliminate these sources
of variation, the variances were estimated from the within-sexes within-litters
sums of squares.

Two similar experiments were done on two separate occasions.
On each occasion the three groups of mice were tested concurrently. In the
second experiment, the hybrids were given a larger dose to compensate for
their greater resistance to the drug. All mice were in the age-range 4-7 weeks,
and those belonging to the same group in the same experiment were always born
within the same eight days. The results of the two experiments were in substantial
agreement, and are set out in the accompanying table.

It will be observed that the variance of the response is about
three and a half times as great in the inbreds as in the hybrids. The difference
is statistically highly significant, as judged by the z-test (P<0.01).
In terms of a hypothetical bioassay experiment, three and a half times as
many of the inbred as of the hybrid mice would have to be used in order to
obtain results of the same accuracy.

More surprisingly, although the "Mousery" mice are
as expected more variable than the hybrids, they are not more variable than
the inbreds. On the contrary, the inbreds are more variable by half as much
again. This difference, although not formally significant, should be regarded
as suggestive, since the variability of the "Mousery" mice was almost
certainly over-estimated in this experiment. It was not known which of these
mice were litter mates. Hence the sum of squares used to estimate their variance
includes a component which was eliminated in the analysis of the other two
groups, namely, that due to differences between litters.

A more clear-cut indication that genetically heterogeneous
random-bred mice may sometimes be less variable than inbred material was obtained
as early as 1939 by Emmens7, who compared the variability of response
to oestrone of a sub-strain of the CBA inbred strain of mice with
that of a random-bred mouse colony. The variance of the inbreds, derived from
the slope of the probit dosage-response curve, was about two and a half times
that of the random-bred mice, and the difference was statistically highly
significant.

Whether random-bred material is more or less uniform in a
given case than inbred will, of course, depend upon circumstances which include
the particular character which is measured and the genetical composition of
the stocks which are compared. As regards the latter, the issue will be largely
decided by the balance between the greater genetic variability among the random-bred
organisms, making for phenotypic variability, and their greater heterozygosity,
making for phenotypic uniformity.

The evidence suggests that the widely adopted policy of using
inbred strains for bio-assay may be a mistaken one, and that inter-strain
F1 hybrids are to be preferred. Such material combines the genetic uniformity
of inbred strains with the 'buffering' action against environmental variations
which heterozygosity exerts.

We are in receipt of a research grant from the Agricultural
Research Council.